1. Field of the Invention
The invention relates to PCR primers designed based on a DNA sequence of a gene encoding malic acid dehydrogenase and a specific DNA of Salmonella typhimurium, to a probe used in PCR, and to a PCR method for the rapid and specific detection of Salmonella typhimurium in food and clinical specimens.
2. Description of related prior art
Among Salmonellae causing food poisoning and Salmonellosis infection, important Salmonellae include S. typhimurium, S. typhi, and S. enteritidis, which play a significant role in main food pathogenic bacteria around the world.
Traditionally, the method for detecting S. typhimurium comprises steps of pre-culturing, culturing on a selective medium, streak culturing and differentiating on a selective agar medium, biochemical identification of suspected colonies, and serological test, which need a time period of at least 5-7 days that might be too late to be of use for understanding of pathogen in a crisis of food poisoning and salmonellosis infection.
Polymerase chain reaction (PCR) can be rapid and reliable for detecting bacteria and virus in various samples. Among all Salmonella serovars, PCR primers useful for detecting S. typhi and S. enteritidis had been reported in literature, while PCR primers for detecting S. typhimurium was rarely seen in literature and patents.
As to the technical level in the state-of-art, related literature and patents can be summarized as follows:
a. Patents associated with the detection of Salmonella: (1) U.S. Pat. No. 5,683,883 (1997) related to PCR primers useful for the detection of all Salmonella serovars: (2) U.S. Pat. No. 5,824,795 (1998) related to PCR detections of S. enteritidis and S. bongori; (3) U.S. Pat. No. 5,714,321 (1998) described nucleotide probes useful for detecting all Salmonella serovars; (4) U.S. Pat. No. 5,804,378 (1988) disclosed nucleotide probes useful for detecting related Salmonella genus; (5) U.S. Pat. No. 5,681,716 (1997) related nucleotide sequences useful for the detection of S. typhi. As stated above, although there were patents relating DNA probe and PCR methods for detecting Salmonella other than S. typhimurium, patent associated with PCR detection of S. typhimurium has been rarely seen. PA1 b. Study reports: (1) Olsen et al (1995) reported oligonucleotide probe useful for the detection of Salmonella and S. typhimurium, which was designed based on the sequence of a cloned 2.3 Kb DNA fragment, however, this probe was used for detecting DNA-DNA hybridization of S. typhimurium; (2) Rahn et al (1992) developed a PCR method for the detection of all Salmonella serovars, which method was based on the sequence of invA gene of S. typhimurium; (3) Cocolin et al (1998) developed PCR method that could detect 33 serotypes of Salmonella; in combination with hydrolytic analysis by restriction enzyme, S. typhimurium could be detected also; (4) Miyamoto et al. (1998) had detected Salmonella including S. typhimurium by utilizing RAPD ; (5) Cohen et al. (1996) devised a PCR method based on the sequence of the finA gene of S. typhimurium for detecting all Salmonella serovars in food samples; (6) Stone et al. (1995) had detected S. typhimurium with a PCR-hybridization method that was not a direct PCR detection method; (7) Tuchili et al. (1995) detected chickens infected by S. gallinarum or S. typhimurium with a PCR method involving the InvA gene, unfortunately, both Salmonella serovars were detected; (8) Nastasiru and Mammina(1995) studied the epidemic S. typhimurium strains by utilizing a PCR-ribotyping process; (9) Way et al. (1993) detected Salmonella, Shigella, E. coli, Citrobacter spp with a multiplex PCR method; (10) in addition, immuno-PCR method had been developed for detecting all serovars Salmonella spp. (Fluit et al 1993; Widjojoatmodjo et al. 1992); (11) Kong et al. (1995) and Chary et al. (1993) reported the detection of S. typhimurium in water by using genes of enterotoxin and Aromatase (ARO-A) of S. typhimurium, however, its specificity was not further confirmed.
Accordingly, there is a need to provide a method for rapid and specific detection of S. typhimurium in food and clinical samples.